Pregnancy status determination in mares using a rapid lateral flow test for measuring serum oestrone sulphate

AIMS: To develop a means of determining pregnancy status in horses based on measuring serum oestrone sulphate (OS) concentrations using a rapid lateral flow immunoassay, and to determine the assay's effectiveness using a visual end-point.

METHODS: Serum samples from mares >100 days post-mating (n=701) were assayed using a nitrocellulose membrane-based lateral flow immunoassay device. The device was developed using membrane-bound 1,3,5 (10)-estratrien-3-ol-17-one conjugated to bovine serum albumin as the capture antigen, and an OS-detection monoclonal antibody coupled to colloidal gold as the visible detection reagent. Concentrations of the coating antigen and OS monoclonal antibody were optimised so that the working range would allow pregnancy status to be determined from a visual end-point. The test was run by adding 0.1 ml serum to the sample well of a plastic cassette encasing the test membrane. As the serum migrated along the membrane, a test dot and control line were generated on it within 5–10 min. The intensity of the test dot was inversely proportional to the concentration of OS in the serum sample being tested. Results were compared with those from a validated OS enzyme immunoassay (EIA) and subsequent foaling or return to oestrus of the mares.

RESULTS: Serum samples with OS concentrations <10 ng/ml, indicative of non-pregnancy in mares >100 days post-mating, generated a test end-point consisting of a highly visible test dot and control line, whereas serum OS concentrations >50 ng/ml, indicative of pregnancy, generated a control line only. The test correctly identified 384/389 (98.7%) non-pregnant mares tested, and 303/312 (97.1%) pregnant mares tested that were >100 days post-mating. The lateral flow test devices were stable for at least 12 months when stored at 4°C, sealed in aluminium pouches with desiccant.

CONCLUSION: This novel, rapid, easy-to-use, lateral flow immunoassay offers a practical alternative to traditional laboratory-based immunoassays for measuring serum OS concentra- tions in mares for determining their pregnancy status.     

Enzymeimmunoassay of oestrone sulphate concentrations in faeces for non-invasive pregnancy determination in mares

AIM: To determine the suitability of measuring faecal oestrone sulphate (OS) by enzymeimmunoassay as a means of determining pregnancy status in mares bred under New Zealand conditions. METHODS: An antibody-coated microtitre plate-based enzymeimmunoassay was used to determine the concentration of OS in faecal and plasma samples obtained from pregnant and non-pregnant mares. RESULTS: In non-pregnant mares, the mean faecal OS concentration was 34 ng/g, and the value three standard deviations above this was 80 ng/g. None of 427 faecal samples collected from 116 non-pregnant mares over a l-year period had an OS concentration >80 ng/g. Only five samples from three mares had an OS concentration >65 ng/g, the value two standard deviations above the mean non-pregnant value. Analysis of faecal OS concentrations in 532 faecal samples collected from 39 pregnant mares showed that as pregnancy progressed, an increasing proportion of faecal samples had OS concentrations >80 ng/g. None of the mares 150 days or more pregnant had faecal OS concentrations <50 ng/g, and 204/220 samples obtained from these mares had faecal OS concentrations >80 ng/g. Following foaling or foetal death, elevated faecal OS concentrations returned quickly to non-pregnant levels. The mean +/- s.e.m. plasma level of OS in five mares bled daily throughout one oestrous cycle was 1.7 +/- 0.2 ng/ml. Sixty-eight blood samples from pregnant mares bled up to five times between 92 days after mating and foaling all had plasma OS concentrations >30 ng/ml, with 64/68 being >50 ng/ml. CONCLUSIONS: This study shows that measuring faecal OS concentrations by enzymeimmunoassay offers a convenient, accurate, non-invasive means of determining pregnancy status in mares from 150 days after mating onwards. Mares with faecal OS concentrations <50 ng/g can be considered not pregnant, while mares with faecal OS concentrations >80 ng/g can be considered pregnant. Those few mares returning a faecal OS concentration between 50 and 80 ng/g should be retested to obtain a conclusive result. Measuring plasma OS concentrations allows pregnancy status to be determined earlier (from 100 days after mating). Moreover, the discrimination between non-pregnant and pregnant levels is greater for OS in plasma than in faeces. CLINICAL RELEVANCE: Measurement of OS concentrations in faeces provides an alternative and non-invasive means of determining pregnancy status in mares from 150 days after mating.     

Attempted conversion of twin to singleton pregnancy in two mares with associated changes in plasma oestrone sulphate concentrations

The removal of one of twin embryos was attempted by infusion of 24% (w/v) saline into the gestation sac in 2 mares by laparotomy. The treatment was successful in one mare (Case 1) and the untreated embryo remained viable. However, neither foetus survived in the second mare (Case 2). Plasma oestrone sulphate (E1S) concentrations fell immediately after treatment in both mares but recovered to approximately 50% of pretreatment levels in Case 1. In Case 2 plasma E1S concentrations declined steadily and were less than 1 ng/ml within 6 days of treatment. These preliminary results suggest that the method may be useful for selective removal of one of twin embryos in mares. Furthermore, plasma E1S concentrations may be a useful indicator of embryonic viability.     

Changes in urinary and plasma oestrone sulphate concentrations after induction of foetal death in mares at 45 days of gestation

Foetal death was induced in 10 Standardbred mares at day 45 of gestation by injecting 20 to 45 ml of hypertonic (24% W/V) saline into the conceptus at surgery. Ten mares underwent sham treatment and acted as controls. Blood and urine samples were collected every other day between days 30 and 45 post ovulation and at 0, 3 and 6 h relative to the infusion of saline in the treated mares, or sham treatment in control mares. Blood and urine samples were then collected daily between days 46 and 55 post ovulation. Urine oestrone sulphate (E1S) concentrations, measured by radioimmunoassay, increased between day 34 and day 36 of gestation in treated and control groups. In mares in which foetal death was induced, urine E1S concentrations declined post-operatively and were significantly (p less than .05) lower than controls by day 50. In plasma, E1S concentrations showed a major increase between days 36 and 40 in both groups. This was followed by a rapid decline after treatment in saline-injected mares, so that by day 48 plasma E1S concentrations in treated mares were significantly (P less than .05) lower than the controls. The results show that urinary and plasma E1S concentrations rise rapidly during early pregnancy, and are associated with a viable foetus after day 45 of pregnancy.     

Changes in serum oestrone sulphate and progesterone levels of red deer hinds during pregnancy

Serum oestrone sulphate (E1S) and progesterone (P) levels were recorded from six red deer hinds throughout the first two-thirds of gestation. E1S was generally undetectable until 30 days post conception when levels rose, peaking between 60 and 80 days, and fell to lower values between 80 and 170 days post conception. P levels rose after conception and remained elevated for the remainder of the study. This study indicates that in general the presence of measurable E1S levels in serum of hinds is associated with pregnancy.     

Latex agglutination test: a simple, rapid and practical method for bovine serum CRP determination.

A semi-quantitative latex agglutination test for bovine serum CRP levels has been established by mixing diluted serum (or diluted standard serum) with a 1% latex suspension containing 0.489 micron latex particles coated with affinity-purified antibody at a ratio of 20 micrograms/mg latex. The agglutination was performed on a glass slide in a moist chamber at room temperature with 45 min. incubation. This test is reliable, reproducible and the results correlate with those of the single radial immunodiffusion (SRID) test. The effect of low temperature storage on CRP concentration revealed a 30% degradation of CRP during 2 years storage at 4 degrees C. The possible role of EDTA addition to prevent a decrease in serum CRP concentration by freezing and thawing is also discussed.     

Evaluation of a rapid reagent strip test for blood glucose determination in diarrheic calves

Sixty-three blood samples from 10 diarrheic calves were tested for glucose concentration by two methods. Plasma glucose concentration was measured by the conventional glucose-6-phosphate dehydrogenase method in the clinical laboratory, and the results compared to those obtained using a rapid reagent strip test for blood glucose concentration measurement. The rapid reagent strip test result could not be used to make an accurate prediction of the actual plasma glucose concentration as determined by the conventional method, due to the wide variability in actual plasma glucose concentrations corresponding to each rapid test result.     

Evaluation of a new early pregnancy test in mares

The goal of this study was to evaluate the accuracy of the early conception factor (ECF) assay test to answer the following questions: (1) Can the test be used reliably to make a decision to flush an embryo donor mare? (2) Can the test be used to detect early embryo loss before day 17 in the mare? (3) Can the test accurately diagnose non-pregnancy?The performance of the test (sensitivity, specificity, positive predictive value [PPV], and negative predictive value [NPV]) was measured against the results of embryo flushing (n = 28) in 1 group and against ultrasound pregnancy diagnosis in another group (n = 17). Serum samples were taken from embryo donor mares on day 8 post-ovulation (day of embryo flushing) and from the embryo transfer recipient mares on days 6 to 8 after embryo transfer (day of pregnancy diagnosis by ultrasound). The tests were set up and read on the same day by 3 blinded observers. The kappa (κ) value, for agreement among the 3 blinded readers of the test, was 0.64.The sensitivity, specificity, PPV, NPV, and accuracy were, respectively, 42.6%, 51.9%, 63.8%, 31.1%, and 46.8%.Under the conditions of the current study, the test proved to be unreliable for detection of the presence of an embryo.     

Validation and method comparison for a point-of-care lateral flow assay measuring equine whole blood insulin concentrations

The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from <139 to >695 pmol/L (<20 to >100 μIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139–278 pmol/L; 20–40 μIU/mL), intermediate (278–417 pmol/L; 40–60 μIU/mL), and high (>417  pmol/L; >60 μIU/mL) concentrations, respectively. For 10 replicates at each level (3 assay lots), inter-assay CVs were 15.9%, 11.0%, and 11.7%, respectively. In the weighted linear regression of 5 measured insulin concentrations against expected concentrations, R² = 0.98, slope = 1.02, and y-intercept = 14.4 pmol/L (2.08 μIU/mL). The Spearman correlation coefficient (r_s) was 0.90 (95% CI: 0.85–0.94) between the WRT and RIA; the WRT = f(RIA) Passing–Bablok regression yielded the fit, y = 1.005x + 24.3 pmol/L (3.50 μIU/mL). The WRT result averaged 10.4% higher than the RIA result, with targeted bias of 25.9, 26.1, and 26.7 pmol/L (3.74, 3.76, and 3.84 μIU/mL) for cutoffs used to diagnose insulin dysregulation of 312, 347, and 451 pmol/L (45, 50, and 65 μIU/mL). Assay clinical sensitivities, specificities, and accuracies determined at the 3 selected clinical cutoffs and using the RIA as gold standard were 87–95%, 92–96%, and 91–95%, respectively (n = 99 samples). Observed total error was 28.4–30.4%. The WRT had acceptable precision, excellent linearity, and good association with the RIA.     

Laparoscopic ovariectomy in mares using a polyamide tie-rap

OBJECTIVE: To describe use of a polyamide tie-rap to ligate the mesovarium during standing laparoscopic ovariectomy in mares. STUDY DESIGN: Prospective study. ANIMALS: Ten mares. METHODS: Bilateral ovariectomy was performed in 10 mares. Standing laparoscopic ovariectomy was performed using 3 portals in the paralumbar fossa. A commercial polyamide tie-rap was prepared as a loop and marked with 4 colored lines close to the buckle, to enable us to check whether the loop was tightened securely. The ovary was grasped with forceps and after the mesovarium was minimally transected cranially and caudally, the loop of the tie-rap was inserted in the abdomen and placed around the mesovarium. It was firmly tightened, until at least 3 of the 4 marks were visible, then the end of the tie-rap was cut. The ovary was transected and removed through an enlarged 3rd portal. The contralateral ovary was removed similarly through the opposite paralumbar fossa. Repeat laparoscopy was performed in 8 mares, 2, 3, 4, and 12 weeks later. RESULTS: None of the mares had postoperative discomfort. On repeat laparoscopy, there was complete encapsulation of the stump and tie-rap after 3-4 weeks. In 2 mares, an adhesion between the left stump and the mesentery of the descending colon was observed. CONCLUSION: Ligation of the mesovarium can be easily and safely performed using a polyamide tie-rap during standing laparoscopic ovariectomy in mares. CLINICAL RELEVANCE: Standing laparoscopic ovariectomy using a polyamide tie-rap is a safe, technically easy and reliable surgical procedure in the mare.     

Accuracy of a rapid enzyme-linked immunosorbent assay to measure progesterone in mares

The aim of this study was to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of progesterone (P4) in mares. Specifically, the objectives were as follows: (1) to determine the specificity and sensitivity of the ELISA test for determination of P4, (2) to measure the potential agreement between the 2 people performing the test, and 3) to evaluate the effect of time on the outcome. Ten mares were sampled on the day before ovulation (D-1), and on days 1 (D1), 3 (D3), and 5 (D5) following ovulation, during the reproductive season. While mares were cycling regularly, estrus was induced by the injection of 5 mg of prostaglandin (PGF2) and monitored starting on the 4th day by daily transrectal palpation and ultrasonography to determine the time of ovulation. Blood was collected and all samples (n=96) were assayed for P4 by a semiquantitative ELISA, by chemiluminescent immunoassay, and by radioimmunoassay (RIA). Based on the RIA, values of P4 on D-1, D1, D3, and D5 were significantly different (P < 0.0001) with mean and standard deviation(s) of 0.004, s = 0.52; 2.05, s = 2.58; 8.37, s = 4.17; and 12.76, s = 4.00 ng/mL respectively. The sensitivity and specificity of the semiquantitative assay were 94% and 95%, respectively for the lowest values of P4 (< 1.0 ng/mL). The value of kappa was 0.90 between 2 individuals performing the test. In conclusion, these results suggest that the semiquantitative test may be used reliably and economically to evaluate P4 levels in equine plasma in the clinical     

Evaluation of a kinetic test kit for the determination of magnesium in bovine serum or plasma

The performance of a new kinetic assay test kit for the determination of magnesium in plasma or serum has been evaluated, using bovine plasma samples. The results obtained were compared with the results from a standard atomic absorption spectrophotometry method and a commercial colorimetric test kit. The kinetic kit compared favourably with the conventional methods and generally gave results with greater precision.